Methods for therapy of neurodegenerative disease of the brain

ABSTRACT

A specific clinical protocol for use toward therapy of defective, diseased and damaged cholinergic neurons in the mammalian brain, of particular usefulness for treatment of neurodegenerative conditions such as Alzheimer&#39;s disease. The protocol is practiced by delivering a definite concentration of recombinant neurotrophin into, or within close proximity of, identified defective, diseased or damaged brain cells. Using a viral vector, the concentration of neurotrophin delivered as part of a neurotrophic composition varies from 10 10  to 10 15  neurotrophin encoding viral particles/ml of composition fluid. Each delivery site receives form 2.5 μl to 25 μl of neurotrophic composition delivered slowly, as in over a period of time ranging upward of 10 minutes/delivery site. Each delivery site is at, or within 500 μm of, a targeted cell, and no more than about 10 mm from another delivery site. Stable in situ neurotrophin expression can be achieved for 12 months, or longer.

RELATED U.S. PATENT APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 11/702,936,filed Feb. 5, 2007 now abandoned, which is a continuation of U.S. patentSer. No. 10/332,306, filed May 9, 2005, which is now abandoned, which isa 371 of PCT/US01/16122, filed May 17, 2001, which is acontinuation-in-part of U.S. patent Ser. No. 09/620,174 filed Jul. 19,2000, now issued as U.S. Pat. No. 6,683,058, which is acontinuation-in-part of U.S. Ser. No. 09/060,543, filed Apr. 15, 1998,now issued as U.S. Pat. No. 6,451,306, the contents of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to methods for treatment of neurodegenerativedisease and methods for delivery of therapeutic neurotrophins into themammalian brain.

HISTORY OF THE RELATED ART

Neurotrophins play a physiological role in the development andregulation of neurons in mammals. In adults, basal forebrain cholinergicneurons, motor neurons and sensory neurons of the CNS retainresponsiveness to neurotrophic factors and can regenerate after loss ordamage in their presence. For this reason, neurotrophins are consideredto have great promise as drugs for the treatment of neurodegenerativeconditions such as Alzheimer's Disease (AD), Parkinson's Disease (PD),amyotrophic lateral sclerosis (ALS), peripheral sensory neuropathies andspinal cord injuries.

Clinical trials for the use of neurotrophins in the treatment of AD, ALSand sensory neuropathies are underway. However, the search for aprotocol for delivery of neurotrophins to target tissues with minimalside effects (e.g., from diffusion to non-targeted cells or immunereaction to the delivery vehicle) and sufficient penetration of the CNS(e.g., bypassing the blood-brain barrier and achieving chronic deliveryof neurotrophin to target cells) has not yet revealed a clear path forclinical administration of neurotrophins. In particular, effectivedelivery methods and dosing parameters have not yet been identified,although several methods have been proposed. Therefore, although theprospects for therapy of neurodegenerative disease of the brain and CNSare believed to be bright, a successful clinical protocol remainselusive,

SUMMARY OF THE INVENTION

The invention provides a clinically useful protocol for delivery ofneurotrophins into the mammalian brain. The invention is particularlyuseful in treating neurodegenerative conditions in primates, in whomneurotrophins delivered according to the invention stimulate growth ofneurons and recovery of neurological function.

More specifically, the invention consists of methods forintraparenchymal delivery of neurotrophins to defective, diseased ordamaged cells in the mammalian brain. In one aspect, the inventionprovides a specific-protocol for use in genetically modifying targetcholinergic neurons (“target cells”) to produce a therapeuticneurotrophin. The genetic modification of target cells is achieved by invivo transfection of neurons targeted for treatment, or by transfectionof cells neighboring these target neurons (neurons or glia), with arecombinant expression vector for expression of the desired neurotrophinin situ.

The location for delivery of individual unit dosages of neurotrophininto the brain is selected for proximity to previously identifieddefective, diseased or damaged target cells in the brain. To intensifyexposure of such target cells to the endogenous growth factors, eachdelivery site is situated no more than about 500 μm from a targeted celland no more than about 10 mm from another delivery site. The totalnumber of sites chosen for delivery of each unit dosage of neurotrophinwill vary with the size of the region to be treated.

Optimally, for delivery of neurotrophin using a viral expression vector,each unit dosage of neurotrophin will comprise 2.5 to 25 μl of anexpression vector composition, wherein the composition includes a viralexpression vector in a pharmaceutically acceptable fluid (“neurotrophiccomposition”) and provides from 10¹⁰ up to 10¹⁵ NGF expressing viralpanicles per ml of neurotrophic composition. According to the method,neurotrophic composition is delivered to each delivery site in the brainby injection through a surgical incision, with delivery to be completedwithin about 5-10 minutes, depending on the volume of neurotrophiccomposition to be provided.

This targeted, regionally specific protocol for nervous system growthfactor delivery avoids limitations imposed by diffusion of substancesacross the blood-brain barrier and through central nervous system (CNS)parenchyma, while avoiding potential adverse effects of neurotrophicfactors delivered intact in a non-directed manner to the CNS.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a reprint of the nucleotide sequence coding for human NT-3 asshown in GENBANK Accession No. E07844 (SEQ ID NO:1).

FIG. 2 is a reprint of the nucleotide sequence coding for human betanerve growth factor as shown in GENBANK Accession No. X52599 (SEQ IDNO'S 2 & 3).

DETAILED DESCRIPTION OF THE INVENTION I. Target Tissues for Treatment ofNeurodegenerative Disorders According to the Invention

The invention identifies and defines the required parameters of a methodfor successful regeneration of neurons in the brain with neurotrophins,especially the neurons whose loss is associated with neurodegenerativeconditions with impairment of cognition such as AD.

The first method parameter defined by the invention is selection of asuitable target tissue. A region of the brain is selected for itsretained responsiveness to neurotrophic factors. In humans, CNS neuronswhich retain responsiveness to neurotrophic factors into adulthoodinclude the cholinergic basal forebrain neurons, the entorhinal corticalneurons, the thalamic neurons, the locus coeruleus neurons, the spinalsensory neurons and the spinal motor neurons. Abnormalities within thecholinergic compartment of this complex network of neurons have beenimplicated in a number of neurodegenerative disorders, including AD,Parkinson's disease, and amyotrophic lateral sclerosis (ALS, also knownas Lou Gehrig's disease). The cholinergic basal forebrain (particularly,the Ch4 region of the basal forebrain) is a particularly suitable targettissue.

Within the primate forebrain, magnocellular neurons Ch1-Ch4 providecholinergic innervation to the cerebral cortex, thalamus and basolateralnucleus of the amygdala. In subjects with neurodegenerative diseasessuch as AD, neurons in the Ch4 region (nucleus basalis of Meynert) whichhave nerve growth factor (NGF) receptors undergo marked atrophy ascompared to normal controls (see, e.g., Kobayashi, et al., Mol. Chem.Neuropathol., 15:193-206 (1991)).

In normal subjects, neurotrophins prevent sympathetic and sensoryneuronal death during development and prevents cholinergic neuronaldegeneration in adult rats and primates (Tuszynski, et al., GeneTherapy, 3:305-314 (1996)). The resulting loss of functioning neurons inthis region of the basal forebrain is believed to be causatively linkedto the cognitive decline experienced by subjects suffering fromneurodegenerative conditions such as AD (Tuszynski, et al., supra and,Lehericy, et al., J. Comp. Neurol., 330:15-31 (1993)).

In human AD, basal forebrain neuronal loss occurs over anintraparenchymal area of approximately 1 cm in diameter. To treataffected neurons over such a large region, treatment with vectorcomposition at upwards of 10 separate in vivo gene vector delivery sitesis desirable. However, in treating localized injuries to the basalforebrain, the affected areas of the brain will likely be smaller suchthat selection of fewer delivery sites (e.g., 5 or fewer) will besufficient for restoration of a clinically significant number ofcholinergic neurons.

Importantly, specific in vivo gene delivery sites are selected so as tocluster in an area of neuronal loss. Such areas may be identifiedclinically using a number of known techniques, including magneticresonance imaging (MRI) and biopsy. In humans, non-invasive, in vivoimaging methods such as MRI will be preferred. Once areas of neuronalloss are identified, delivery sites are selected for stereotaxicdistribution so each unit dosage of NGF is delivered into the brain at,or within 500 μm from, a targeted cell, and no more than about 10 mmfrom another delivery site.

II. Dosing Requirements and Delivery Protocol for Treatment ofNeurodegenerative Disorders According to the Invention

A further parameter defined by the invention is the dosage ofneurotrophin to be delivered into the target tissue. In this regard,“unit dosage” refers generally to the concentration of neurotrophin/mlof neurotrophic composition. For viral vectors, the neurotrophinconcentration is denned by the number of viral particles/ml ofneurotrophic composition. Optimally, for delivery of neurotrophin usinga viral expression vector, each unit dosage of neurotrophin willcomprise 2.5 to 25 μl of a neurotrophic composition, wherein thecomposition includes a viral expression vector in a pharmaceuticallyacceptable fluid and provides from 10¹⁰ up to 10¹⁵ NGF expressing viralparticles per ml of neurotrophic composition.

The neurotrophic composition is delivered to each delivery cell site inthe target tissue by microinjection, infusion, scrape loading,electroporation or other means suitable to directly deliver thecomposition directly into the delivery site tissue through a surgicalincision. The delivery is accomplished slowly, such as over a period ofabout 5-10 minutes (depending on the total volume of neurotrophiccomposition to be delivered).

Those of skill in the art will appreciate that the direct deliverymethod employed by the invention obviates a limiting risk factorassociated with in vivo gene therapy; to with, the potential fortransfection of non-targeted cells with the vector carrying the NGFencoding transgene. In the invention, delivery is direct and thedelivery sites are chosen so diffusion of secreted NGF takes place overa controlled and pre-determined region of the brain to optimize contactwith targeted neurons, while minimizing contact with non-targeted cells.

Startingly, in primates, a viral vector (AAV) with an operableneurotrophin encoding transgene has been shown to express humanneurotrophin after delivery to the brain and to the CNS for up to 12months. As such, the invention provides a chronically available sourcefor neurotrophin in the brain.

III. Materials for Use in Practicing the Invention

Materials useful in the methods of the invention include in vivocompatible recombinant expression vectors, packaging cell lines, helpercell lines, synthetic in vivo gene therapy vectors, regulatable geneexpression systems, encapsulation materials, pharmaceutically acceptablecarriers and polynucleotides coding for nervous system growth factors ofinterest.

A. Neurotrophins

Known nervous system growth factors include nerve growth factor (NGF),brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3),neurotrophin-4/5(NT-4/5), neurotrophin-6 (NT-6), ciliary neurotrophicfactor (CNTF), glial cell line-derived neurotrophic factor (GDNF), thefibroblast growth factor family (FGF's 1-15), leukemia inhibitory factor(LIF), certain members of the insulin-like growth factor family (e.g.,IGF-1), the neurturins, persephin, the bone morphogenic proteins (BMPs),the immunophilins, the transforming growth factor (TGF) family of growthfactors, the neuregulins, epidermal growth factor (EGF),platelet-derived growth factor (PDGF), and others. NGF and NT-3 inparticular have been tested with promising results in clinical trialsand animal studies (see, e.g., Hefti and Weiner, Ann. Neurol.,20:275-281 (1986); Tuszynki and Gage, Ann. Neurol., 30:625-636 (1991);Tuszynski, et al., Gene Therapy, 3:305-314 (1996) and Blesch andTuszynski, Clin. Neurosci., 3:268-274 (1996)). Of the known nervoussystem growth factors, NGF and NT-3 (for treatment of the CM region, asin AD) are preferred for use in the invention.

Human (h) NGF and hNT3 are preferred for use in therapy of human diseaseaccording to the invention due to their relatively low immunogenicity ascompared to allogenic growth factors. However, other nervous systemgrowth factors are known which may also be suitable for use in theinvention with adequate testing of the kind described herein.

Coding polynucleotides for hNGF and hNT3 are known, as are codingsequences for neurotrophins of other mammalian species (e.g., mouse, inwhich the coding sequence for NGF is highly homologous to the humancoding sequence). For example, a cDNA including the coding sequence forhNGF is reported in GenBank at E03015 (Kazuo, et al., Japanese PatentApplication No. JP 19911175976-A, while the nucleotide sequence ofgenomic hNGF (with putative amino acid sequence) is reported in GenBankat HSBNGF (Ullrich, Nature, 303:821-825 (1983)) and the mRNA sequence isreported in GenBank at HSBNGFAC (Borsani, et al., Nucleic Acids Res.,18:4020 (1990)). The genomic nucleotide sequence of hNT3 is reported inGenBank at E07844 (Asae, et al., JP Patent Application No.1994189770-A4). These references are incorporated herein to illustrateknowledge in the art concerning nucleotide and amino acid sequences foruse in synthesis of neurotrophins. Exemplary reprints of nucleotidesequences coding for NGF and NT-3 obtained from the GENBANK nucleotidedatabase are provided in, respectively, FIGS. 1 and 2.

B. Recombinant Expression Vectors

The strategy for transferring genes into target cells in vivo includesthe following basic steps: (1) selection of an appropriate trans gene ortrans genes whose expression is correlated with CNS disease ordysfunction; (2) selection and development of suitable and efficientvectors for gene transfer, (3) demonstration that in vivo transductionof target cells and transgene expression occurs stably and efficiently;(4) demonstration that the in vivo gene therapy procedure causes noserious deleterious effects; and (5) demonstration of a desiredphenotypic effect in the host animal.

Although other vectors may be used, preferred vectors for use in themethods of the present invention are viral and non-viral vectors. Thevector selected should meet the following criteria: 1) the vector mustbe able to infect targeted cells and thus viral vectors having anappropriate host range must be selected; 2) the transferred gene shouldbe capable of persisting and being expressed in a cell for an extendedperiod of time (without causing cell death) for stable maintenance andexpression in the cell; and 3) the vector should do little, if any,damage to target cells.

Because adult mammalian brain cells are non-dividing, the recombinantexpression vector chosen must be able to transfect and be expressed innon-dividing cells. At present, vectors known to have this capabilityinclude DNA viruses such as adenoviruses, adeno-associated virus (AAV),and certain RNA viruses such as HIV-based lentiviruses and felineimmunodeficiency virus (FIV). Other vectors with this capability includeherpes simplex virus (HSV).

For example, a HIV-based lentiviral vector has recently been developedwhich, like other retroviruses, can insert a transgene into the nucleusof host cells (enhancing the stability of expression) but, unlike otherretroviruses, can make the insertion into the nucleus of non-dividingcells. This lentiviral vector has been shown to stably transfect braincells after direct injection, and stably express a foreign trans genewithout detectable pathogenesis from viral proteins (see, Naldini, etal., Science, 272:263-267 (1996), the disclosure of which isincorporated by reference). Following the teachings of the researcherswho first constructed the HIV-1 retroviral vector, those of ordinaryskill in the art will be able to construct lentiviral vectors suitablefor use in the methods of the invention (for more general referenceconcerning retrovirus construction, see, e.g., Kriegler, Gene Transferand Expression, A Laboratory Manual, W. Freeman Co. (NY 1990) andMurray, E J, ed., Methods in Molecular Biology, Vol. 7, Humana Press (NJ1991)).

Adenoviruses and AAV have been shown to be quite safe for in vivo useand have been shown to result in long-term gene expression in vivo; theyare therefore preferred choices for use in the methods of the invention,where safety and long-term expression of nervous system growth encodingtransgenes (persisting for longer than necessary to stimulate regrowthof injured or diseased neurons) is necessary. Those of ordinary skill inthe art are familiar with the techniques used to construct adenoviraland AAV vectors and can readily employ them to produce vectorcompositions useful in the claimed invention (for reference, see, e.g.,Straus, The Adenovirus, Plenum Press (NY 1934), pp. 451-496; Rosenfeld,et al., Science, 252:431-434 (1991); U.S. Pat. No. 5,707,618 [adenovirusvectors for use in gene therapy]; and U.S. Pat. No. 5,637,456 [methodfor determining the amount of functionally active adenovirus in a vectorstock], the contents of each of which is incorporated herein toillustrate the level of skill in the art).

Lentiviral-based vectors such as HIV and FIV are currently at earlierstages of development but also are attractive candidates for in vivogene therapy based upon stability of expression in vivo and safetyprofiles.

Herpes viruses, alpha viruses and pox viruses are alsowell-characterized virus vectors which may be applied to the methods ofthe invention. Of these vectors, adeno-associated vectors are anespecially attractive choice for their lack of pathogenicity and abilityto insert a transgene into a host genome.

Non-viral delivery methods are also an option for use in the methods ofthe invention. In particular, the plasmid (in a “naked” orlipid-complexed form), lipoplexes (liposome complexed nucleic acids),amino acid polymer complexes with nucleic acids and artificialchromosomes are all non-viral gene delivery agents which aredemonstrably able to transduce cells and deliver a foreign transgene.Synthetic in vivo' gene therapy vectors are also an option for use inthe methods of the invention.

Construction of vectors for recombinant expression of nervous systemgrowth factors for use in the invention may be accomplished usingconventional techniques which do not require detailed explanation to oneof ordinary skill in the art. For review, however, those of ordinaryskill may wish to consult Maniatis et al., in Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, (NY 1982).

Briefly, construction of recombinant expression vectors employs standardligation techniques. For analysis to confirm correct sequences invectors constructed, the ligation mixtures may be used to transform ahost cell and successful transformants selected by antibiotic resistancewhere appropriate. Vectors from the transformants are prepared, analyzedby restriction and/or sequenced by, for example, the method of Messing,et al., (Nucleic Acids Res., 9:309, 1981), the method of Maxam, et al.,(Methods in Enzymology, 65:499, 1980), or other suitable methods whichwill be known to those skilled in the art. Size separation of cleavedfragments is performed using conventional gel electrophoresis asdescribed, for example, by Maniatis, et al., (Molecular Cloning, pp.133-134, 1982).

Expression of a gene is controlled at the transcription, translation orpost-translation levels. Transcription initiation is an early andcritical event in gene expression. This depends on the promoter andenhancer sequences and is influenced by specific cellular factors thatinteract with these sequences. The transcriptional unit of manyprokaryotic genes consists of the promoter and in some cases enhancer orregulator elements (Banerji et al., Cell 27:299 (1981); Corden et al.,Science 209:1406 (1980); and Breathnach and Chambon, Ann. Rev. Biochem.50:349 (1981)). For retroviruses, control elements involved in thereplication of the retroviral genome reside in the long terminal repeat(LTR) (Weiss et al., eds., The molecular biology of tumor viruses: RNAtumor viruses, Cold Spring Harbor Laboratory, (NY 1982)). Moloney murineleukemia virus (MLV) and Rous sarcoma virus (RSV) LTRs contain promoterand enhancer sequences (Jolly et al., Nucleic Acids Res. 11:1855 (1983);Capecchi et al., In: Enhancer and eukaryotic gene expression, Gulzmanand Shenk, eds., pp. 101-102, Cold Spring Harbor Laboratories (NY 1991).Other potent promoters include those derived from cytomegalovirus (CMV)and other wild-type viral promoters.

Promoter and enhancer regions of a number of non-viral promoters havealso been described (Schmidt et al., Nature 314:285 (1985); Rossi and deCrombrugghe, Proc. Natl. Acad. Sci. USA 84:5590-5594 (1987)). Methodsfor maintaining and increasing expression of transgenes in quiescentcells include the use of promoters including collagen type I (1 and 2)(Prockop and Kivirikko, N. Eng. J. Med. 311:376 (1984); Smith and Niles,Biochem. 19:1820 (1980); de Wet et al., J. Biol. Chem., 258:14385(1983)), SV40 and LTR promoters.

In addition to using viral and non-viral promoters to drive transgeneexpression, an enhancer sequence may be used to increase the level oftransgene expression. Enhancers can increase the transcriptionalactivity not only of their native gene but also of some foreign genes(Armelor, Proc. Natl. Acad. Sci. USA 70:2702 (1973)). For example, inthe present invention collagen enhancer sequences are used with thecollagen promoter 2(I) to increase transgene expression. In addition,the enhancer element found in SV40 viruses may be used to increasetransgene expression. This enhancer sequence consists of a 72 base pairrepeat as described by Gruss et al., Proc. Natl. Acad. Sci. USA 78: 943(1981); Benoist and Chambon, Nature 290:304 (1981), and Fromm and Berg,J. Mol. Appl. Genetics, 1:457 (1982), all of which are incorporated byreference herein. This repeat sequence can increase the transcription ofmany different viral and cellular genes when it is present in serieswith various promoters (Moreau et al., Nucleic Acids Res. 9:6047 (1981).

Transgene expression may also be increased for long term stableexpression using cytokines to modulate promoter activity. Severalcytokines have been reported to modulate the expression of transgenefrom collagen 2(I) and LTR promoters (Chua et al., connective TissueRes., 25:161-170 (1990); Elias et al., Annals N.Y. Acad. Sci.,580:233-244 (1990)); Seliger et al., J. Immunol. 141:2138-2144 (1988)and Seliger et al, J. Virology 62:619-621 (1988)). For example,transforming growth factor (TGF), interleukin (IL)-1, and interferon(INF) down regulate the expression of transgenes driven by variouspromoters such as LTR. Tumor necrosis factor (TNF) and TGF1 up regulate,and may be used to control, expression of transgenes driven by apromoter. Other cytokines that may prove useful include basic fibroblastgrowth factor (bFGF) and epidermal growth factor (EGF).

Collagen promoter with the collagen enhancer sequence (Coll(E)) can alsobe used to increase transgene expression by suppressing further anyimmune response to the vector which may be generated in a treated brainnotwithstanding its immune-protected status. In addition,anti-inflammatory agents including steroids, for example dexamethasone,may be administered to the treated host immediately after vectorcomposition delivery and continued, preferably, until anycytokine-mediated inflammatory response subsides. An immunosuppressionagent such as cyclosporin may also be administered to reduce theproduction of interferons, which downregulates LTR promoter and Coil(E)promoter-enhancer, and reduces transgene expression.

C. Pharmaceutical Preparations

To form a neurotrophic composition for use in the invention,neurotrophin encoding expression vectors (including, without limitation,viral and non-viral vectors) may be placed into a pharmaceuticallyacceptable suspension, solution or emulsion. Suitable mediums includesaline and liposomal preparations.

More specifically, pharmaceutically acceptable carriers may includesterile aqueous of non-aqueous solutions, suspensions, and emulsions.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishes, electrolytereplenishes (such as those based on Ringer's dextrose), and the like.

Preservatives and other additives may also be present such as, forexample, antimicrobials, antioxidants, chelating agents, and inert gasesand the like. Further, a composition of neurotrophin trans genes may belyophilized using means well known in the art, for subsequentreconstitution and use according to the invention.

A colloidal dispersion system may also be used for targeted genedelivery Colloidal dispersion systems include macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes.Liposomes are artificial membrane vesicles which are useful as deliveryvehicles in vitro and in vivo. It has been shown that large unilamellarvesicles (LUV), which range in size from 0.2-4.0 μm can encapsulate asubstantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within theaqueous interior and be delivered to cells in a biologically active form(Fraley, et al., Trends Biochem. Sci., 6:77, 1981). In addition tomammalian cells, liposomes have been used for delivery of operativelyencoding transgenes in plant, yeast and bacterial cells. In order for aliposome to be an efficient gene transfer vehicle, the followingcharacteristics should be present: (1) encapsulation of the genesencoding the antisense polynucleotides at high efficiency while notcompromising their biological activity; (2) preferential and substantialbinding to a target cell in comparison to non-target cells; (3) deliveryof the aqueous contents of the vesicle to the target cell cytoplasm athigh efficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosohatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted gene delivery system may be modified in avariety of ways. In the case of a liposomal targeted delivery system,lipid groups can be incorporated into the lipid bilayer of the liposomein order to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

IV. Methods for Delivery of Vector Composition

Following the protocol defined by the invention, direct delivery of aneurotrophic composition may be achieved by means familiar to those ofskill in the art, including microinjection through a surgical incision(see, e.g., Capecchi, Cell. 22:479-488 (1980)); electropotation (see,e.g., Andreason and Evans, Biotechniques, 6:650-660 (1988)); infusion,chemical complexation with a targeting molecule or co-precipitant (e.g.,liposome, calcium), and microparticle bombardment of the target tissue(Tang, et al, Nature, 356:152-154 (1992)).

V. Animal Models and Clinical Evaluation

In non-human primate subjects (Example III), the process of agingsimulates the neurological changes in the brain experienced in aginghumans. An non-aged animal model that also mimics loss of cholinergicneurons in, for example, AD, is transection of the fomix pathwayconnecting the septum from the hippocampus, which causes spontaneousdegeneration of the same neurons which degenerate through aging (see,e.g., Example II). In rats and primates, such transections causeretrograde degeneration of cholinergic and non-cholinergic cell bodiesin the septal nucleus and nucleus basalis (Ch4 region) of the brain.

These animals are tractable to treatment with neurotrophins, and modelclinical responsiveness to such treatment comparable to aged humans(especially the non-human primates, whose brains are most similar insize and structure to humans). Data demonstrating the use and efficacyof the methods of the invention in these animal models are provided inthe Examples.

Clinical evaluation and monitoring of treatment can be performed usingthe in vivo imaging techniques described above as well as through biopsyand histological analysis of treated tissue. In the latter respect,basal forebrain cholinergic neuronal numbers can be quantified in atissue sample using, for example, anti-neurotrophin antibody (forimmunoassay of secreted neurotrophin) or NGF-receptor (p75) and cholineacetyltransferase (ChAT) for labeling of neurons. A sample protocol forin vitro histological analysis of treated and control tissue samples isdescribed in the Examples.

The invention having been fully described, examples illustrating itspractice are set forth below. These examples should not, however, beconsidered to limit the scope of the invention, which is defined by theappended claims. Those of ordinary skill in the art will appreciate thatwhile the Examples illustrate an ex vivo application of the invention,the results achieved will be accessible through in vivo delivery of thenervous system growth factor encoding trans genes described, as taughtherein, with in vivo gene delivery sites and direct delivery meanssubstituted for the grafting sites and grafting methods discussed in theExamples.

In the examples, the abbreviation “min.” refers to minutes, “hrs” and“h” refer to hours, and measurement units (such as “ml”)—are referred toby standard abbreviations. All printed materials cited are incorporatedherein by reference.

Example I Adeno-Associated Virus Vector Construction and Viral ParticleProduction

For adeno-associated viral vector construction, an expression cassettewas cloned containing the following elements: 1) cytomegaloviruspromoter (CMV_(ie)); 2) a multiple cloning site; 3) an internal ribosomeentry site followed by the coding sequence for the active, B-sequence ofhuman nerve growth factor (NGF) or the enhanced form of greenfluorescent protein (EGFP); and, 4) a SV40 polyadenylation sequence.

The complete cassette was cloned into the vector psub201 (American TypeCulture Collection) after XbaI digestion to remove the AAV codingsequences. For NGF expression the coding sequence for human NGF (see,GENBANK Accession No. X52599) was inserted into the multiple cloningsite of psub-CXIE resulting in the vector psub-CXIE-NGF. This vector,termed psub-CXIE, was used to prepare control GFP expressing virusparticles. Thus, this vector was used for the production of particlescoding for .NGF and GFP.

Recombinant adeno-associated virus was produced by co-transfection of 18μg expression plasmid psub-CXIE or psub-CXIE-NGF, 18 μg pXX2 and 54 μgpXX6 per 150 mm plate of subconfluent 293 cells. Transfected cells wereharvested 48 h later and adeno-associated virus was purified bylodixanol density gradient centrifugation and Heparin affinitychromatography. For virus concentration and buffer exchanges Biomax™100K filters were used. Aliquots of virus were stored at −80 C. Thenumber of viral particles was determined using Southern dot blotting.

Example II In Vivo Gene Transfer in an Animal Model of Cholinergic CellDeath

Animals received injections of adeno-associated viral (AAV) vector intoan in vivo rat model of cholinergic cell death, to determine the extentand parameters of AAV-NGF vector delivery to prevent neuronaldegeneration using in vivo gene delivery. To prepare the animal modeladult Fischer 344 rats underwent formix transections to induce basalforebrain cholinergic neuronal death. NGF-AAV vector (CXIE-NGF) orcontrol, EGFP-AAV vector (CXIE) was injected into the cholinergic basalforebrain at a range of 2.5 to 10 μl of stock vector solution containingfrom 10¹⁰-10¹² particles per ml (neurotrophic composition). Particleswere injected over a time period of 3-5 min. into the right hemisphereat the following coordinates: AP-0.3; ML-0.5; DV-6 from brain surface.The skin was closed and animals were allowed to survive for 2-4 weeks.

AAV vector delivery induced increasing zones of transfection withincreasing concentration and volume of vector panicles. Maximal levelsof in vivo gene expression were achieved at the highest concentration ofvector and highest volume of injection. Over the two week time period ofthis experiment, persistent in vivo gene expression was demonstrated.Gene expression was primarily manifested in neurons (>90%) as opposed toglia. No adverse effects of the injections were evident.

Thus, vector doses of 2.5 to 10 μl vector stock at a range of 10¹⁰-10¹²particles per ml were well-tolerated, resulted in optimal vectordelivery to the host cholinergic neuronal system, and did not result inadverse events or undesired vector spread beyond the target neuronalnucleus. NGF and enhanced GFP expression were evident for at least twoweeks in vivo.

Example III Model of Alzheimer's Disease Through Aging in Primates

Twelve aged and four adult non-aged Macaca mulatta (rhesus) monkeys wereexperimental subjects. Non-aged animals (n=4, mean age=9.64±1.90 yrs)did not undergo surgical procedures and their intact brains werestudied. Aged monkeys were divided into two experimental groups: NGFrecipients (n=6, mean age=22.55±0.56 yrs) and control subjects (n=6,mean age=23.51±1.07 yrs). All procedures and animal care adheredstrictly to NTH, AAALAC, USDA, Society for Neuroscience, and internalinstitutional guidelines (of the University of California, San Diego)for experimental animal health, safety and comfort.

Example IV Preparation of h-NGF Secreting Fibroblasts

To demonstrate responsiveness to NGF, aged monkeys receivedintraparenchymal grafts of autologous fibroblasts genetically modifiedto produce and secrete human NGF, as previously described. Briefly,autologous fibroblasts obtained from skin biopsies were geneticallymodified in vitro to produce and secrete the active portion of humanNGF. Transduction procedures were carried out usingreplication-incompetent retroviral vectors derived from Moloney murineleukemia virus (MLV). Transduced cells were selected by growth in theneomycin analog G418.

Production of biologically active NGF was verified by induction ofneurite outgrowth from PC 12 cells as described; production of NGF mRNAwas determined by Northern blot; and amounts of NGF produced from cellswere assayed by NGF ELISA specific for human NGF and sensitive to 5pg/ml. Optimal NGF-producing bulk clones were amplified to numberssufficient for in vivo grafting by serial passaging. Cells wereharvested by gentle trypsinization for in vivo grafting.

Example V Intraparenchymal Delivery into Primates of FibroblastsGenetically Modified to Produce h-NGF

Monkeys underwent pre-operative MRI scans (see, Tuszynski, et al., GeneTherapy, 3:305-314, 1996) to visualize basal forebrain target graftingregions (see, Mesulam et al., J. Comp. Neurol, 214:170-197, 1983). Aftergenerating stereotaxic grafting coordinates from MRI scans, each monkeyreceived intraparenchymal grafts of autologous NGF-secretingfibroblasts.

Stereotactic coordinates for surgery were generated from magneticresonance images (MR) of the brain of each subject. The rostral andcaudal boundaries of Ch4 were identified on each subject's MR scan,making reference to primate histological brain sections and to standardprimate brain atlases. The total rostral-caudal distance of Ch4 wasmeasured on the MR scan, and five graft injection sites were chosen thatwere equally distributed over this rostral-caudal distance.

The sites for desired ventral-dorsal (VD) and medial-lateral (ML)injections were chosen such that cell grafts were deposited just dorsalto the desired target at each coordinate (within 500 urn), and exactlycentered in the medrolateral (ML) plane at the maximal density ofcholinergic neuronal somata (estimated by review of histologicalsections at the corresponding AP level). Thus, five grafts weredeposited on each side of the Ch4 region per subject, or ten totalgrafts per subject. Real-time coordinates for in vivo injections werecalculated from calibration scales on the MR image. Subjects underwentsurgical grafting in the same stereotaxic apparatus that MR scans wereperformed in.

To place the grafts, animals were placed into a primate stereotaxicapparatus and a midline scalp incision was used to expose the skull. TheAP and ML stereotaxic coordinates for the BFC system were used to definethe margins of the craniotomy site. Following craniotomy, a ML zeroreference point was obtained by measuring the midpoint of the superiorsagittal sinus. The dura was incised and reflected to expose the pialsurface. The pial surface at each injection site was used as a VD zeroreference point for that injection site.

Using the zero reference points obtained in the AP, ML, and VD planesand the stereotaxic injection coordinates calculated from that animal'sMR scan, 5 μl of cells were injected into each of 5 sites over therostral-caudal extent of the Ch4 targeted region bilaterally (10 graftstotal per animal) using 25-gauge Hamilton syringe. Grafts were generallytargeted to a position slightly dorsal to but within 500 μm of Ch4nuclei. The injection rate was controlled at 5 μl/mm. Cells wereinjected at a concentration of 1.0×10⁵ cells/μl (for a total of 10million grafted cells per animal), a concentration that optimallymaintains ceils in suspension without clumping but sufficientlyconcentrated to maximize number of surviving cells in vivo. Monkeyssurvived for three months before sacrifice.

Some control aged subjects received intraparenchymal grafts as notedabove. These grafted cells consisted either of autologous fibroblaststransduced to express the reporter gene beta-galactosidase (n=6monkeys). Beta-gal production was assessed in vitro using a specificanti-beta-gal antibody. Cells were grafted into intraparenchymal sitesin numbers identical to those described above for NGF graft recipients.

For all surgical procedures, primates were preanesthetized with 25 mg/kgketamine IM. They were then anesthetized with isoflurane administered byendotracheal intubation. Post-operatively animals were closelymonitored, and received supportive care and appropriate analgesics whenindicated. Animals were placed in the same primate stereotaxic apparatus(Crist Instruments) that was used to perform MRI scans. A midline scalpincision exposed the skull. A 2.5×5 cm sagittally oriented craniotomywas performed on each side of the hemicranium, and the dura was incisedand reflected to expose sites for stereotaxically guided ceilinjections. Ten μl of cells were injected into each site through a 25ga. Hamilton syringe at a rate of 1 μl/minute. Postoperatively, allexperimental subjects were observed closely for signs of discomfort ortoxicity. After a three-month survival-period, animals were perfusedtranscardially for one hour with a 4% solution of paraformaldehyde in0.1 M phosphate buffer followed by 5. % sucrose solution in the samebuffer for 20 minutes. The brain was stereotaxically blocked in thecoronal plane.

Example VI Reversal of Age-Related p75 Expression Loss

In AD brains, NGF accumulates in regions of basal forebrain cholinergicneurons and is decreased in the basal forebrain, leading to thehypothesis that insufficient retrograde transport of NGF promotes thedegeneration of basal forebrain cholinergic neurons observed in AD. Inhumans, basal forebrain cholinergic neuron dysfunction has been closelylinked with age-related cognitive and memory impairment.

In the mammalian brain, it is believed that the p75 receptorcollaborates with the TrkA receptor to form high-affinity binding sitesfor NGF. Although activation of TrkA is sufficient for NGF to rescueaxotomized cholinergic neurons, disruption of NGF binding to p75 reducesNGF binding to TrkA. Hence, co-expression of the two receptors can leadto greater responsiveness to NGF. Conversely, loss of expression maylead to decreased responsiveness to NGF. Expression of both p75 and TrkAis regulated by NGF, so that a loss of NGF signalling further reducesthe amount of both p75 and TrkA. Combined with a loss of expression ofTrkA in AD brains, leading to reduced amounts of TrkA. protein in boththe basal forebrain and the cortex, decreased p75 expression maycontribute to a decline in retrograde NGF signalling. Thus, p75expression is a marker for NGF binding, basal forebrain cholinergicneuron dysfunction and cognitive impairment.

To determine the effect of the method of the invention on p75 expressionin treated primate brains, monkeys were treated as described in ExampleIII. Each subject was then deeply anesthetized with ketamine andnembutal and perfused transcardially for 1 hour with a 4% solution ofparaformaldehyde in 0.1M phosphate buffer, followed by 5% sucrosesolution in the same buffer for 20 min. The brains were thenstereotaxically blocked in the coronal plane to obtain a single blockcontaining the full AP extent of Ch4.

Coronal sections were cut on a freezing microtome set at 40 urn. Everysixth section was processed for p75 immunoreactivity. Briefly, sectionswere washed thoroughly in Tris-buffered saline (TBS) and endogenousperoxidases were quenched by incubating in a 0.6% hydrogen peroxidesolution. Sections were rinsed in TBS and then blocked using 5% donkeyserum with 0.5% Triton X-100 in TBS (TBS++). Incubation in primaryantibody (monoclonal diluted 1:100 in TBS++) occurred for 24 hours atroom temperature. Sections were rinsed in TBS++, incubated in secondaryantibody (biotinylated donkey-anti-mouse diluted 1:500 in TBS++) for 1hour, rinsed again in TBS++, and then incubated for 90 minutes using aVector ABC kit. p75-labeled neurons were then visualized usingdiaminobenzidine (DAB) as a chromogen. Sections were then mounted andcoverslipped.

p75-labeled neurons were quantified in Ch4i neurons using stereologicalprocedures. Ch4i was targeted, in this study since this region is theprincipal site of origin of cholinergic projections to conical regionsthat modulate memory.

Ch4 can be divided topographically into three subdivisions, the anterior(Ch4a), intermediate (Ch4i), and posterior (Ch4p). The anteriorsubdivision is further divided into medial (Ch4 am) and lateral (Ch4al)sectors, which are divided by a vascular structure or rarefication inthe density of neurons. However, as Ch4a travels in the posteriordirection toward Ch4i, the division between Ch4 am and Ch4al becomesless distinct and in some disappears. In this region the ansapeduncularis, the characteristic structure of Ch4i, begins to make itsappearance, The ansa peduncularis divides Ch4i into ventral (Ch4iv) anddorsal (Ch4id) components. There is typically also a portion of theanterior commissure present over the lateral portion of Ch4id at thislevel that serves as the anterior boundary of Ch4i. At the posteriorboundary of Ch4i, Ch4iv and Ch4id merge into a single nucleus embeddedin the intersection of the globus pallidus, putamen, and optic tract.

Stereological counts were performed on every sixth section through theentire extent of Ch4i. The NeuroZoom™ stereology computer programrunning on an Apple Macintosh PowerPC™ and connected to a Javelin™ videocamera mounted on an Olympus Vanox™ HBT-3 microscope was used to conductstereology by the well-known West optical dissector method. Briefly, theregion of interest (Ch4i) was outlined in NeuroZoom using a 1×objective. Specific stereology parameters were then set in NeuroZoom asfollows:

Fraction (percent of area): 5%

Counting frame size: x=66.46 um, y=53.73 um

Section thickness: 40 um

These parameters were adjusted to minimize the coefficient of error ofthe estimate (CE(P)) while maximizing the efficiency of sampling.

The NeuroZoom program controlled movement from one counting frame to thenext by moving a Ludl motorized stage mounted on the microscope. Ch4ineurons were counted using a 60× high numerical aperture (1.40) oilobjective. Cells were marked to be included in the count if they met thefollowing criteria: 1) they were p75-labeled; 2) the soma was within thecounting frame (or touching the inclusion boundary) but did not touchthe exclusion boundary; 3) a clearly visible nucleus was present; and 4)the nucleus was best in focus within the inclusion volume (i.e., the top12.5% and bottom 12.5% were excluded, and the nucleus was not in focusin either of these exclusion volumes). Multiple group comparisons weremade by analysis of variance (ANOVA) with post-hoc analysis usingFisher's least squares difference.

The number of p75-labeled Ch4i neurons was compared between four groupsof rhesus monkeys, two of which were unoperated and two of whichreceived intraparenchymal grafts of genetically-modified fibroblasts.Young monkeys (mean age=9.375±1.058) constituted one of the unoperatedgroups, while aged monkeys (mean age=25.139±2.455) comprised the otherunoperated group. Of the two aged groups which received grafts to thebasal forebrain, one (mean age=22.639+0.463) received grafts of cellsmodified to produce and secrete NGF, and the other (meanage=23.321+0.927) received grafts of cells modified to produce andsecrete beta-gal.

There were significantly fewer p75-labeled neurons in Ch4i fromunoperated aged monkeys than from unoperated young monkeys (p<0.01). Themean number of p75-labeled Ch4i neurons from NGF-grafted aged monkeyswas significantly greater than from control-grafted aged monkeys(p<0.04). Further, there number of p75-labeled Ch4i neurons inNGF-grafted aged monkeys did not differ from numbers in unoperated youngmonkey brains (p=0.1288).

These results demonstrate that there is spontaneous loss of expressionof the low-affinity neurotrophin receptor (p75) in cholinergic neuronsin the basal forebrain, and that re-expression of p75 can be induced byintraparenchmal delivery of NGF.

Example VII Histology Confirming In Vivo Uptake of Transgene Expressionof NGF and Lack of Beta-Amyloid Induction

Sections of brain tissue after humane sacrifice of the test animals werecut at 40 μm intervals on a freezing microtome. Every sixth section wasprocessed for Nissl stain or hematoxylin and eosin. Immunocytochemicallabeling against amyloid was performed using an amyloid-specificmonoclonal antibody (anti-A4). Sections lacking primary antibody wereprocessed to verify specificity of labeling. A representative sectionper subject was quantified from each of the following regions: temporal,frontal, cingulate, insular, parietal and occipital cortices; amygdalaand hippocampus; and the intermediate division of the Ch4 region(Nucleus Basalis of Meynert). Sampled sections from each subject wereclosely matched in region and size. The total number of amyloid plaquesper region was quantified and recorded. Observers were blinded to theidentity of the tissue being quantified.

All grafted subjects showed surviving cell grafts within 500 um of eachgrafting site. There was no qualitative difference in fibroblastmorphology and overall graft size between NGF— and control-graftrecipients. Grafts were most frequently located adjacent to theintermediate division of the Ch4 region of the basal forebrain, but inall cases included at least one graft located within the anterior andposterior divisions of the Ch4 region.

No amyloid plaques at all were detected in adult, non-aged primatetissue. In contrast, control aged monkeys showed a significant increasein amyloid immunolabeling in the frontal, temporal insular and cingulatecortices and amygdala, and extremely small increases in the parietalcortex and hippocampus relative to non-aged monkeys. No plaques at allwere present in the cholinergic basal forebrain in any group.

In aged control animals, plaques typically showed a dense central coreand a less dense surrounding halo of immunoreactive deposition product,an appearance typical of “mature” plaques observed in AD. Thisimmunolabeling pattern is consistent with previous reports in agedprimate brain. However, no increase in amyloid labeling was observed inthe aged, NGF-grafted brains, indicating that three months ofintraparenchymal NGF delivery does not increase beta-amyloid plaquedeposition in the aged primate brain. Thus, the benefits of NGF graftingin the brains of primates exhibiting AD symptoms can be achieved withoutrisk of stimulating amyloid deposition in response to the graft trauma.

Initially, group differences were statistically determined by analysisof variance, with post-hoc analysis utilizing Fisher's least squaredifference. However, since non-aged adult monkeys showed no amyloidplaques, comparisons between NGF-treated and control aged monkeys weremade using unpaired two-way student's t-test.

The invention claimed is:
 1. A method for delivery of a neurotrophin totargeted defective, diseased or damaged neurons in the mammalian brain,the method comprising directly delivering fibroblasts geneticallymodified to express the neurotrophin into one or more delivery siteswithin an intraparenchymal region of the brain containing targetedneurons; wherein the genetically modified fibroblasts are depositedwithin 500 um from a targeted cell, and no more than about 10 mm fromanother delivery site; and wherein further contact between the targetedcell and the expressed neurotrophin ameliorates the defect, disease ordamage in said targeted neurons.
 2. The method according to claim 1wherein the delivery sites are within the Ch4 region of the cholinergicbasal forebrain.
 3. The method according to claim 1 wherein theameliorated disease is Parkinson's disease.